Myosin Va (myoVa) and myosin VI (myoVI) are double-headed, processive molecular motors that transport intracellular cargo over long distances by way of actin filament tracks. With their ability to travel in opposite directions along the polarized actin cytoskeleton, myoVa transport is critical for exocytosis while myoVI is associated with endocytosis. To successfully deliver cargo, both myoVa and myoVI must overcome physical challenges presented by the intracellular milieu and the interactions with other motors that are attached to the same cargo. To assess these motors' inherent transport capabilities, we will use state-of-the- art single molecule biophysical techniques with high spatial (> 6nm) and temporal (<2ms) resolution to characterize the stepping dynamics of single myoVa or myoVI motors, independently or when linked together in a tug of war. Aim #1 will determine how the individual heads of a myoVa molecule coordinate and adjust their stepping behavior to maintain processive motion in response to both resistive and assistive forces. This will be accomplished by monitoring the individual heads that are labeled with different color quantum dots (Qdots) as force is applied to the motor in a combination laser trap-TIRF microscope. In Aim #2, myoVI will be similarly characterized. However, myoVI is unique in that it takes unexpectedly large steps, challenging the swinging lever arm model. Through structural mutagenesis, we will determine whether the proximal and/or medial tail serve as surrogate lever arms to allow myoVI to step processively. Finally in Aim #3, we will build complexity in vitro by linking myoVa and myoVI motors to the same Qdot cargo, as a model for intracellular cargo transport by multiple motors. We will unambiguously determine the stepping dynamics of both motors simultaneously as they attempt to transport the same cargo. With the data gathered in Aims #1 and #2, we will quantitatively model and predict the outcome of these tug of war scenarios as it relates to intracellular bidirectional transport by ensembles of molecular motors.